Project Description The overall goal of this project is to elucidate the structure and function of histone acetyltransferase (HAT) enzymes. These proteins use the acetyl-CoA cofactor to acetylate the nitrogen of specific lysine residues within the N-terminal tails of core histones and work in concert with other histone modification enzymes to provide a mechanistic link between chromatin alteration and gene activation. HAT proteins are found from yeast to man and regulate genes involved in several different biological processes including cell cycle progression, dosage compensation and hormone signaling. Aberrant HAT function has also been correlated with human disease, including leukemic translocations involving the CBP and MOZ HATs, and p300 HAT alterations in colorectal and gastric cancers. HAT proteins fall into subfamilies with characteristic substrate specificity for histones, and in some cases non-histone substrates. Over the last funding periods, we have used X-ray crystallography and complementary biochemical and enzymatic analysis to characterize the substrate binding and catalytic properties of the Gcn5/PCAF, MYST and most recently the p300/CBP subfamilies of HAT proteins. Our results demonstrate that while these different HAT families have a structurally conserved central enzymatic core, they employ different catalytic mechanisms. We also show that the regions within the HAT domain that flank the catalytic core within these enzymes have divergent structure and function and are thus particularly important in imparting different transcriptional regulatory properties to these HAT enzymes. We will now continue to use a combination of structure, biochemistry and enzymology to carry out additional studies on the MYST and p300/CBP family of HATs and to extend our studies to 2 other acetyltransferases, an evolutionarily ancient archaeal HAT called PAT and a newly characterized yeast HAT called Rtt109 that cooperates with the histone chaperone protein Vps75 to acetylate K56 of histone H3 to promote genomic stability. Specifically, the four aims of the proposal are to (1) Study the mechanism of p300/CBP regulation by an autoregulatory loop and by the ATF2 transcription factor, and screen for p300/CBP inhibitors; (2) Characterize the mechanism for acetylation, substrate recognition and autoregulation by the archaeal PAT acetyltransferase; (3) Characterize the mechanism for acetylation by the Rtt109 HAT and its modulation by the Vps75 histone chaperone; and (4) Investigate the catalytic mechanism of the MYST HATs and characterize the structure and function of the heterotrimeric Sas2/4/5 complex. Together, these studies will provide new molecular insights into the substrate specificity and catalytic mechanism of histone acetyltransferases, and how these activities are modulated by intra-auto-regulatory domains and other associated protein factors. Public Health Relevance Statement Histone Acetyltranferase (HAT) enzymes play key roles in regulating gene expression and aberrant HAT function has also been correlated with several human diseases, including leukemic translocations involving the CBP and MOZ HATs, and p300 HAT alterations in colorectal and gastric cancers. The overall goal of this project is to elucidate the structure, chemistry and function of histone acetyltransferase (HAT) enzymes and to use this information to aid in the design small molecule drugs to treat HAT-associated diseases.